From the journal RSC Chemical Biology Peer review history

06-Jun-2021

Dear Dr Garcia:

Manuscript ID: CB-ART-05-2021-000099
TITLE: Alkyl vs Aryl Modular Modification on mitochondrial uptake on triphenylphosphonium delivery vectors: A comparative analysis

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary. In particular providing quantitative measurements for the mitochondrial uptake followed with calculated ratio between mitochondrial and cellular uptake to demonstrate the mitochondrial selectivity and addressing the reviewer comment on voltage-driven translocation of triphenylphosphonium ions through the lipid membrane.

Please submit a revised manuscript which addresses all of the reviewers’ comments. Further peer review of your revised manuscript may be needed. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link:

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Supporting our community through Covid-19
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The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

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Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

************

Reviewer 1

This manuscript aims at comparing various effects of alkyl vs aryl modification on TPP in terms of mitochondrial uptake, cytotoxicity, lipophilicity, membrane penetration simulation, and so on. Although the issues on TPP-based mitochondria-targeted systems are very important, the comparison is rarely studied. Also, the study was well-designed, well-written, and well-organized. Nevertheless, some additional results should be performed to support current results.

1. Mitochondrial membrane potential is strongly related to mitochondrial uptake of TPP-based molecules. Thus, mitochondrial membrane potentials of all listed TPP derivatives should be monitored in terms of time-dependent or time-independent manners.
2. Although some confocal results explained the mitochondrial uptake effects of TPP derivatives, the quantitative study on mitochondrial uptake should be carried out. After isolating mitochondria, the amount of TPP-based derivatives in the isolated mitochondria should be monitored by a flow cytometry.
3. Mitochondrial selectivity is very important. Thus, the relative uptake ratio of mitochondrial uptake to cellular uptake should be calculated.

Reviewer 2

This is an interesting paper contributing some good physical measurements and experiments in cells. These show how different distributions of alkyl groups on triarylphosphonium cations affect these cations' lipophilicity and their behaviour in cells. Such lipophilic cations are widely used for targeting compounds to the mitochondria so a better understanding of their behaviour is important. The measurements themselves are carried out correctly, but the interpretation needs to be improved before publication.

Major points:

Firstly, although the IC50 can correlate with the degree of uptake of a lipophilic cation into the mitochondrial matrix in some cases, toxicity can arise from a variety of effects. For example, the very different shapes of isomers 1c and 3b will affect how they interact with and cause curvature in the membrane. This section would be much better framed as a comparison of toxicity rather than evidence for uptake. Uptake could be directly measured in isolated mitochondria using an ion-specific electrode or by HPLC, but I think there is enough new information here without such additional work. Please can the authors simply rewrite this section as a comparison of toxicity.

The manuscript provides useful insight into how well different triarylphosphonium salts cross membranes using measured and calculated logPs, which is important to understanding their biological properties. However, the reason that lipophilic cations are used to target the mitochondrial matrix is that they accumulate 100-1000-fold driven by the large membrane potential across the inner mitochondrial membrane (120-180 mV, negative inside). This involves the movement of charge across the membrane and so requires the translocation of the cation alone. It cannot be satisfactorily modelled using partitioning of the neutral salt.

Rokitskaya et al (Phys. Chem. Chem. Phys., 2019, 21, 23355-23363) have experimentally interrogated voltage-driven translocation of triaryldodecylphosphonium ions including analogues of 1 (n = 11) and 2 (n = 11) by measuring current across black lipid membranes. Rokitskaya et al also confirmed that such movement was best modelled by the transfer energy for a cation (rather than salt) moving from the aqueous phase to the non-polar alkane as originally described by Robb et al, reference 45. The computational models and measurements presented in the current manuscript are for salts, not lipophilic cations. The authors should modify their text to include this important distinction, citing Rokitskaya and giving some explanation of how their work on bulk partitioning complements the studies and models of Rokitskaya and Robb on the transfer of cations. They also need to reframe their discussion of the modelling in Figure S5. Unlike the work of Robb et al, Figure S5 deals with both ions crossing together in different ways, not the difference between a lipophilic cation crossing alone without an anion and a lipophilic cation crossing with its counterion.

These points apart, the work is a valuable contribution.

Minor points: The text is rather long and the discussion of the characterisation of simple phosphonium salts in the main text is not needed and should be transferred to the supplementary information. There are a few spelling and grammar points that could easily be corrected in a revised manuscript e.g. "alchemical" on page 3. I also found the phrase on page 1 "comparative analysis of alkyl versus aryl modular modification" ambiguous. The paper addresses the effect of distributing alkyl substituents differently

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.
Referee 1
Comments to the Author
This manuscript aims at comparing various effects of alkyl vs aryl modification on TPP in terms of mitochondrial uptake, cytotoxicity, lipophilicity, membrane penetration simulation, and so on. Although the issues on TPP-based mitochondria-targeted systems are very important, the comparison is rarely studied. Also, the study was well-designed, well-written, and well-organised. Nevertheless, some additional results should be performed to support current results.
1. Mitochondrial membrane potential is strongly related to mitochondrial uptake of TPP-based molecules. Thus, mitochondrial membrane potentials of all listed TPP derivatives should be monitored in terms of time-dependent or time-independent manners.
ANSWER: To address this point, we have carried out additional experiments to monitor the mitochondrial membrane potential after treatment of HeLa cells with all nine TPP+ derivatives, 1a – 3c. The mitochondrial membrane potential (MMP) was assessed using the JC-1 dye and quantified with a microplate reader. The detailed experimental procedures are shown below. In short, HeLa cells were treated with 2.5 µM of 1a – 3c for four hours, followed by JC-1 staining. The 2.5 µM incubation concentration was selected as it lies in the middle of the IC50 ranges previously determined (ca. 25 - 0.25 µM). All experiments were performed in triplicate, and the ratio of J-aggregate (red) to monomer (green) is presented below (relative to untreated control).
The results indicate that treatment with 1a – 3c resulted in a significant impact on the MMP. (Figure 1, blue) For highly lipophilic salts (2c, 3b, 3c), a drastic decrease in the MMP was observed, resulting in a R/G ratio similar to FCCP treatment. On the other hand, salts with low lipophilicity (1a, 1b, 2a) showed membrane hyperpolarisation, possibly due to the inhibition of ATPase or specific protein complexes.1-2 The results from the MMP assay correlated well with logP, with an evident sigmoidal relationship between MMP and logP (Figure A3). Interestingly, clustering of 1c, 2b and 3a (isomers) in the plot was observed as well. These observations indicate a possible competition between multiple interactions affecting the MMP (ATPase inhibition, proton leak, etc), but additional studies will be required to determine the exact mechanism. Included in the ESI – Figure S4 (pages S10). Highlighted in yellow.
To differentiate the MMP depolarisation with cytotoxic effects, a cell viability assay was performed concurrently, and the results are shown below (Figure A2). Despite the large decrease of the MMP, cell viability at this time for all nine compounds remain above 80%, with the lowest cell viability in 2c at 84%. This further supports that the MMP is indeed affected by the TPP+ salts. Included in the ESI – Figure S3 (pages S10). Highlighted in yellow.
An additional experiment was performed to investigate the reversibility of this effect. After 4 hours of incubation with 1a – 3c, the medium was aspirated and replaced with fresh DMEM. After an additional 4 hours of incubation, the JC-1 and cell viability assays were performed (Figure A1, red). The JC-1 assay revealed that the R/G ratio of most compounds, with the exception of the high lipophilicity derivatives, converged towards 1.2x the untreated control, showing a reduction in the magnitude of the hyper/depolarising effects seen previously. This is presumably due to a low residual amount of TPP+ still present after washing. Cell viability of all nine salts remained relatively similar compared to the previous data, indicating that further cell death was insignificant. Included in the manuscript as Figure 3 (page 3) and ESI Figure S3 (page S10). Highlighted in yellow.

In summary, the MMP was affected from incubation with all TPP+ salts, which were known to induce proton leaks and affect mitochondrial respiration.3,4 The effects appear to be correlated with the lipophilicity of the nine salts, with no clear difference between alkyl chain extension and aryl methylation. Removal of TPP+ from the cell medium results in a slow restoration of the MMP, with no further cell death observed. However, the detailed mechanism of these effects will be difficult to ascertain without further detailed studies.
These studies have been included in the manuscript (Figure 3, page 3) and ESI (Figures S2-S4, pages 9-10) – Highlighted in yellow.

Figure A1. R/G ratio after treatment with compounds 1a – 3c and FCCP, before washing (blue) and after washing (red). Error bars refer to the standard deviations. -

Figure A2 . Cell viability after treatment with compounds 1a – 3c and FCCP, before washing (blue) and after washing (red). Error bars refer to the standard deviations.

Figure A3. Correlation between R/G ratio and logP. Error bars refer to the standard deviations.
Experimental procedure
Experiments were performed in triplicate. 3 x 104 HeLa cells were seeded in a 96 well plate and allowed to attach overnight. The cells were treated with an addition of 11 µL of 10x stock solutions of 1a – 3c (25 µM) and FCCP (1 mM). Untreated controls and background samples were included. The plate was allowed to incubate at 37°C in 5% CO2 for 4 hours. 100 µL of JC-1 or resazurin solution were layered on top of the cells, to a final working concentration of 1 µM and 0.02 mg mL-1, respectively. The plate was allowed to incubate for an additional 1 hour. The cells treated with JC-1 was washed twice with phosphate-buffered saline, and the fluorescence signals were measured using a microplate reader. The following excitation/emission wavelengths were used : JC-1 aggregate = 535 nm/590 nm, JC-1 monomer = 475 nm/530 nm, resazurin/resorufin = 560 nm/590 nm. Included in the ESI, page 9 – Highlighted in yellow.
[1] M. Forkink, G. R. Manjeri, D. C. Liemburg-Apers, E. Nibbeling, M. Blanchard, A. Wojtala, J. A. M. Smeitink, M. R. Wieckowski, P. H. G. M. Willems and W. J. H. Koopman, Biochim. Biophys. Acta, Bioenerg., 2014, 1837, 1247-1256.
[2] D. G. Nicholls and S. L. Budd, Physiol. Rev., 2000, 80, 315-360.
[3] C. Reily, T. Mitchell, B. K. Chacko, G. A. Benavides, M. P. Murphy and V. M. Darley-Usmar, Redox. Biol., 2013, 1, 86-93.
[4] C. A. Kulkarni, B. D. Fink, B. E. Gibbs, P. R. Chheda, M. Wu, W. I. Sivitz and R. J. Kerns, J. Med. Chem., 2021, 64, 662-676.
Additional references were included in the main text as reference 39 – 42. (Page 7) – Highlighted in yellow.
2. Although some confocal results explained the mitochondrial uptake effects of TPP derivatives, the quantitative study on mitochondrial uptake should be carried out. After isolating mitochondria, the amount of TPP-based derivatives in the isolated mitochondria should be monitored by a flow cytometry.
ANSWER: Flow cytometry was conducted on isolated mitochondria after incubating with dyes with the longest chain length (4c) and highest degree of methylation (4e). Relative to the untreated control, an increase in fluorescence intensity was observed for both compounds 4c and 4e, indicating the uptake of both dyes. A higher uptake of 4c relative to 4e was observed, which was consistent with the trends observed from the confocal imaging experiments. The experimental results (Figure A4) and detailed experimental procedure are shown below.
These results have been included in the ESI (Page 11) – Highlighted in yellow.
Experimental procedure
2 x 106 HeLa cells in 2 mL of DMEM w/o phenol red supplemented with 10% FBS were harvested and incubated in 100 nM of 4c and 4e at 37°C for 1 hour. Cells were washed with phosphate-buffered saline, and were subsequently lysed via needle homogenisation (20 strokes, 30G). The mitochondria isolation was done using a mitochondria isolation kit (Miltenyi Biotech, 130-094-532), and the isolated mitochondria was kept under ice. Isolated mitochondria samples were analysed using a BD LSRFortessa X-20 flow cytometer. Included in the ESI (Page 11) – Highlighted in yellow

Figure A4. Histogram of fluorescence intensity for compound 4c, 4e and the control. Included in the ESI – Figure S5 (pages S11). Highlighted in yellow.

3. Mitochondrial selectivity is very important. Thus, the relative uptake ratio of mitochondrial uptake to cellular uptake should be calculated.
ANSWER: The mitochondrial selectivity of these compounds are well known, whether from chain length increase or from methylation.1-4 While we acknowledge that the quantitative information on subcellular distribution within the cell would be invaluable, it would be highly time-consuming to obtain. Hence, in order to provide a more quantitative picture of mitochondrial selectivity, we have performed statistical analysis on confocal imaging data, and the Van Steensel’s cross correlation function (CCF) curve, colocalisation cytofluorogram, Pearson’s coefficient, Mander’s coefficient (M1 and M2, Ch1 = MitoTracker, Ch2 = Fluorescein) were calculated from the confocal images between the newly synthesised dyes and MitoTracker. The statistical analyses were performed (n = 5) using ImageJ and the JACoP plugin. The values obtained are shown below:
Compound n Pearson’s coefficient M1 M2
4a 5 0.568 ± 0.108 0.954 ± 0.023 0.874 ± 0.013
4b 5 0.884 ± 0.006 0.956 ± 0.013 0.967 ± 0.007
4c 5 0.896 ± 0.006 0.973 ± 0.004 0.974 ± 0.003
4d 5 0.856 ± 0.021 0.956 ± 0.012 0.965 ± 0.004
4e 5 0.877 ± 0.018 0.954 ± 0.007 0.967 ± 0.006

Compounds 4b – 4e exhibits a high degree of colocalisation with MitoTracker, with high Pearson’s coefficient from 0.856 to 0.896. Although compound 4a has a relatively low r value of 0.568, the Van Steensel’s curve supports that it too localises in the mitochondria, with the maximum in the cross-correlation function at dx = 0 for all compounds. The high Mander’s coefficient, M1 and M2, which ranges from 0.954 to 0.974 for compounds 4b – 4e indicates the bulk of the fluorescence signal overlaps between the MitoTracker and fluorescein channels, further supporting a high degree of mitochondrial selectivity.
These results have been included into the manuscript (Table 2 on page 6) and the ESI (pages 11-12) – Highlighted in yellow 
Compound Cytofluorogram Van Steensel’s CCF
4a
4b
4c
4d
4e

[1] T. I. Rokitskaya, M. P. Murphy, V. P. Skulachev and Y. N. Antonenko, Bioelectrochemistry, 2016, 111, 23-30.
[2] Y. N. Antonenko, S. S. Denisov, D. N. Silachev, L. S. Khailova, S. S. Jankauskas, T. I. Rokitskaya, T. I. Danilina, E. A. Kotova, G. A. Korshunova, E. Y. Plotnikov and D. B. Zorov, Biochim Biophys Acta, 2016, 1860, 2463-2473.
[3] A. J. Smith, P. J. Gawne, M. T. Ma, P. J. Blower, R. Southworth and N. J. Long, Dalton Transactions, 2018, 47, 15448-15457.
[4] Z. Hu, Y. Sim, O. L. Kon, W. H. Ng, A. J. Ribeiro, M. J. Ramos, P. A. Fernandes, R. Ganguly, B. Xing, F. Garcia and E. K. Yeow, Bioconjug Chem, 2017, 28, 590-599.
Referee 2
Comments to the Author
This is an interesting paper contributing some good physical measurements and experiments in cells. These show how different distributions of alkyl groups on triarylphosphonium cations affect these cations' lipophilicity and their behaviour in cells. Such lipophilic cations are widely used for targeting compounds to the mitochondria so a better understanding of their behaviour is important. The measurements themselves are carried out correctly, but the interpretation needs to be improved before publication.
Major points:
Firstly, although the IC50 can correlate with the degree of uptake of a lipophilic cation into the mitochondrial matrix in some cases, toxicity can arise from a variety of effects. For example, the very different shapes of isomers 1c and 3b will affect how they interact with and cause curvature in the membrane. This section would be much better framed as a comparison of toxicity rather than evidence for uptake. Uptake could be directly measured in isolated mitochondria using an ion-specific electrode or by HPLC, but I think there is enough new information here without such additional work. Please can the authors simply rewrite this section as a comparison of toxicity.
ANSWER: The relevant sentence has been edited. Instead of “For a rapid screening of mitochondrial uptake, the 72-hour cell viability assay was conducted using HeLa cells by the resazurin reduction assay.”, it has been replaced by “To evaluate the cytotoxicity of 1a – 3c, the 72-hour cell viability assay was conducted using HeLa cells by the resazurin reduction assay.”- On page 2 – Highlighted in yellow
The following sentence has also been omitted from the updated manuscript: “Thus, the dose-response curves of these compounds should reveal the mitochondrial accumulation of the compounds, as a higher accumulation of the TPP+ salts should result in a greater cytotoxicity.”
The manuscript provides useful insight into how well different triarylphosphonium salts cross membranes using measured and calculated logPs, which is important to understanding their biological properties. However, the reason that lipophilic cations are used to target the mitochondrial matrix is that they accumulate 100-1000-fold driven by the large membrane potential across the inner mitochondrial membrane (120-180 mV, negative inside). This involves the movement of charge across the membrane and so requires the translocation of the cation alone. It cannot be satisfactorily modelled using partitioning of the neutral salt.
Rokitskaya et al (Phys. Chem. Chem. Phys., 2019, 21, 23355-23363) have experimentally interrogated voltage-driven translocation of triaryldodecylphosphonium ions including analogues of 1 (n = 11) and 2 (n = 11) by measuring current across black lipid membranes. Rokitskaya et al also confirmed that such movement was best modelled by the transfer energy for a cation (rather than salt) moving from the aqueous phase to the non-polar alkane as originally described by Robb et al, reference 45. The computational models and measurements presented in the current manuscript are for salts, not lipophilic cations. The authors should modify their text to include this important distinction, citing Rokitskaya and giving some explanation of how their work on bulk partitioning complements the studies and models of Rokitskaya and Robb on the transfer of cations. They also need to reframe their discussion of the modelling in Figure S5. Unlike the work of Robb et al, Figure S5 deals with both ions crossing together in different ways, not the difference between a lipophilic cation crossing alone without an anion and a lipophilic cation crossing with its counterion.
ANSWER: The experimental logP measured does indeed reflect the property of the neutral salt and is not purely the cation component. It is precisely due to these limitations that computational studies are required to study the cation without external influences. However, there appears to be a misunderstanding with regards to this experiment. Although we have a neutral system, with the lipophilic cations plus chloride ions, only the cations crossed the membrane in most experiments. We simulated a cation crossing together with its ion pair (both ions close to each other) in a single case (Figure S5 – now Figure S9). We concluded that the crossing of the cation alone was the most favourable. The experiment depicted on Figure S5 (now Figure S9) as highlighted by the reviewer is the only case where a restraint is applied to TPP – chloride distances to restrict membrane transport to occur as an ion-pair, in order to evaluate whether ion-pairing could improve membrane transport for our systems. Without any restraints applied to TPP – chloride distances, the number of chloride contacts during the translocation process was approximately zero at the middle of the bilayer, indicating that transport of the ion occurred as a lipophilic cation, rather than an ion pair. Therefore, we calculated "the difference between a lipophilic cation crossing alone without an anion and a lipophilic cation crossing with its counterion" and not "both ions crossing together in different ways".
Nevertheless, we agree that our simulations and calculations could have some contribution of the salt, and experimentally this could be more difficult to discriminate, but we are essentially simulating the translocation of the cations and their lipophilicity. Overall, we think that our conclusions agree with previous theoretical results, and we hope these new modifications also answer the reviewer’s comments. We have revised the manuscript to make this important distinction more clear. The following discussion was added to the manuscript:
We observed that ion pairing should not be favourable for the translocation of this cation, which is in agreement with existing literature for TPMP using DFT-based continuum model calculations by Hartley, Murphy et al., which have shown that the energetic penalty for TPMP to be transferred from water to the non-aqueous hexane phase as an ion-pair was larger than for the TPMP cation alone.50 Although we the same partition was obtained with or without constraining interatomic distances, the free energy barrier of 1a without constraints were lower by ca. 2 kcal∙mol-1. On page 5 – Highlighted in yellow
We also made minor changes to the electronic supporting information:
“In Figure S9, we compare the free energy profiles of 1a and of 1a when we restrained the distance between the cation and one Cl- counter-ion (which we define as 1a ion-pair).” On page S21 – Highlighted in yellow
And the caption of Figure S5 (now S9):
“Figure S9. Free energy profiles of the translocation of 1a and of 1a with a harmonic potential restrain for the distance between the cation and a Cl- counter-ion (defined as 1a ion-pair), and average number of contacts with Cl- ions in a POPC hydrated bilayer model system. The top two panels show a representation of the hydrated bilayer model system and the partial density profiles for the different functional groups or molecules in the system. The third panel shows the free energy profiles for the translocation of 1a and of 1a ion-pair (black and green, respectively). The bottom panel shows the average number of contacts with Cl- counter-ions in the simulation cell for both situations (considering a distance threshold of 0.6 nm). Vertical lines define the four-membrane regions as described in the main text.” On page S21 – Highlighted in yellow
The following reference highlighted by the reviewer has been added as reference 49: T. I. Rokitskaya, V. B. Luzhkov, G. A. Korshunova, V. N. Tashlitsky and Y. N. Antonenko, Phys. Chem. Chem. Phys., 2019, 21, 23355-23363. On page 8 – Highlighted in yellow
These points apart, the work is a valuable contribution.
Minor points: The text is rather long and the discussion of the characterisation of simple phosphonium salts in the main text is not needed and should be transferred to the supplementary information. There are a few spelling and grammar points that could easily be corrected in a revised manuscript e.g. "alchemical" on page 3. I also found the phrase on page 1 "comparative analysis of alkyl versus aryl modular modification" ambiguous. The paper addresses the effect of distributing alkyl substituents differently
Point 1. We have reduced the discussion on the characterisation as suggested by the reviewer
Point 2. “Alchemical” is a standard adjective for calculations that transform a molecule into another by deleting/creating atoms.
Point 3. The sentence has been updated to: “Herein we report the first study comparing the effects of alkyl vs aryl modification on mitochondrial uptake for triphenylphosphonium moieties (Figure 1).” On page 1 – Highlighted in yellow

05-Aug-2021

Dear Dr Garcia:

Manuscript ID: CB-ART-05-2021-000099.R1
TITLE: Alkyl vs Aryl Modular Modification on mitochondrial uptake on triphenylphosphonium delivery vectors: A comparative analysis

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after including minor revisions in the text as suggested by the reviewer.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link :

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***

https://mc.manuscriptcentral.com/rsccb?link_removed

(This link goes straight to your account, without the need to log in to the system. For your account security you should not share this link with others.)

Alternatively, you can login to your account (https://mc.manuscriptcentral.com/rsccb) where you will need your case-sensitive USER ID and password.

You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.

Supporting our community through Covid-19
While our publishing services are running as usual, we also know that this is a very challenging time for everyone, for many different reasons. If any aspect of the publishing process is worrying you – for example you think you may struggle to meet a pre-determined deadline – please let us know, and we will work out an answer together.

RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy from CASRAI, https://casrai.org/credit/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines http://www.rsc.org/journals-books-databases/journal-authors-reviewers/author-responsibilities/ for more information.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

************

Reviewer 2

This is a very nice paper. The changes are all good, but a couple of small modifications are needed: the sentence "The logPmem obtained were also found to be well-correlated with experimental logP," which describes Rokitskaya's work (reference 49) should be modified to read "The logPmem obtained were also found to be well-correlated with experimental logP and consistent with translocation rate constants in black lipid membranes, " so that the reader is aware of the existence of the rate constant data. This will also strengthen the paper as a useful summary of all relevant work. The "we" in "Although we the same partition was obtained with or without.." should be deleted.

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.

Referee 1
Comments to the Author
This is a very nice paper. The changes are all good, but a couple of small modifications are needed: the sentence "The logPmem obtained were also found to be well-correlated with experimental logP," which describes Rokitskaya's work (reference 49) should be modified to read "The logPmem obtained were also found to be well-correlated with experimental logP and consistent with translocation rate constants in black lipid membranes, " so that the reader is aware of the existence of the rate constant data. This will also strengthen the paper as a useful summary of all relevant work. The "we" in "Although we the same partition was obtained with or without.." should be deleted.
ANSWER:
We have modified "The logPmem obtained were also found to be well-correlated with experimental logP,[49]" to "The logPmem obtained were also found to be well-correlated with experimental logP , and consistent with previously reported translocation rate constant trends in black lipid membranes,[49]" We have slightly modified the sentence suggested by the referee to avoid confusion by directly correlating thermodynamic (i.e., logP) with kinetic (i.e., membrane translocation rates) measurements. The modified sentence is highlighted in yellow – page 5.
The "we" in "Although we the same partition was obtained with or without.." has been deleted. The modified sentence is highlighted in yellow – page 5.

21-Aug-2021

Dear Dr Garcia:

Manuscript ID: CB-ART-05-2021-000099.R2
TITLE: Alkyl vs Aryl Modular Modification on mitochondrial uptake on triphenylphosphonium delivery vectors: A comparative analysis

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Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology


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